1. Field of the Invention
The present invention relates to a procedure for the production of enantiomerically pure monomeric and dimeric C-10 non-acetal derivatives of natural trioxane artemisinin having high in vitro antimalarial, antiproliferative and antitumor activities. The present invention further relates to the formation of a novel trioxane aldehyde compound produced via a chemoselective Cxe2x80x94C bond formation at the C-10 position upon reaction of artemisinin trioxane lactone with lithiothiazole or lithiobenzothiazole. This trioxane aldehyde may then be reacted with organolithium, Grignard, and phosphorus ylide nucleophiles exclusively via carbonyl addition.
2. Description of the State of Art
Each year approximately 200-300 million people experience a malarial illness and over 1 million individuals die. In patients with severe and complicated disease, the mortality rate is between 20 and 50%.
Plasmodium is the genus of protozoan parasites which is responsible for all cases of malaria and Plasmodium falciparum is the species of parasite that is responsible for the vast majority of fatal malaria infections. Malaria has traditionally been treated with quinolines such as chloroquine, quinine, mefloquine, and primaquine and with antifolates such as sulfadoxine-pyrimethamine. Unfortunately, most P. falciparum strains have now become resistant to chloroquine, and some, such as those in Southeast Asia, have also developed resistance to mefloquine and halofantrine; multidrug resistance is developing in Africa also.
The endoperoxides are a promising class of antimalarial drugs which may meet the dual challenges posed by drug-resistant parasites and the rapid progression of malarial illness. The first generation endoperoxides include artemisinin and several synthetic derivatives, discussed in further detail below.
Artemisia annua L., also known as qinghao or sweet wormwood, is a pervasive weed that has been used for many centuries in Chinese traditional medicine as a treatment for fever and malaria. Its earliest mention, for use in hemorrhoids, occurs in the Recipes for 52 Kinds of Diseases found in the Mawangdui Han dynasty tomb dating from 168 B.C. Nearly five hundred years later Ge Hong wrote the Zhou Hou Bei Ji Fang (Handbook of Prescriptions for Emergency Treatments) in which he advised that a water extract of qinghao was effective at reducing fevers. In 1596, Li Shizhen, the famous herbalist, wrote that chills and fever of malaria can be combated by qinghao preparations. Finally, in 1972, Chinese chemists isolated from the leafy portions of the plant the substance responsible for its reputed medicinal action. This crystalline compound, called qinghaosu, also referred to as QHS or artemisinin, is a sesquiterpene lactone with an internal peroxide linkage.
Artemisinin is a member of the amorphane subgroup of cadinenes and has the following structure (I). 
Artemisinin or QHS was the subject of a 1979 study conducted by the Qinghaosu Antimalarial Coordinating Research Group involving the treatment of 2099 cases of malaria (P. vivax and P. falciparum in a ratio of about 3:1) with different dosage forms of QHS, leading to the clinical cure of all patients. See, Qinghaosu Antimalarial Coordinating Research Group, Chin. Med. J., 92:811 (1979). Since that time artemisinin has been used successfully in many thousand malaria patients throughout the world including those infected with both chloroquine-sensitive and chloroquine-resistant strains of P. falciparum. Assay of artemisinin against P. falciparum in vitro revealed that its potency is comparable to that of chloroquine in two Hanian strains (Z. Ye, et al., J. Trad. Chin. Med., 3:95 (1983)) and of mefloquine in the Camp (chloroquine-susceptible) and Smith (chloroquine-resistant) strains, D. L. Klayman, et al., J. Nat. Prod., 47:715 (1984).
Although artemisinin is effective at suppressing the parasitemias of P. vivax and P. falciparum, the problems encountered with recrudescence, and the compound""s insolubility in water, led scientists to modify artemisinin chemically, a difficult task because of the chemical reactivity of the peroxide linkage which is believed to be an essential moiety for antimalarial activity.
Reduction of artemisinin in the presence of sodium borohydride results in the production of dihydroartemisinin (II-1) or DHQHS, (illustrated in structure II below), in which the lactone group is converted to a lactol (hemiacetal) function, with properties similar to artemisinin. Artemisinin in methanol is reduced with sodium borohydride to an equilibrium mixture of xcex94-and E-isomers of dihydroartemisinin. The yield under controlled conditions is 79% (artemisinin, 0.85M with NaBH4 6:34M, 7:5 equivalents in methanol, 12 L at 0-5xc2x0 C. for about 3 hours followed by quenching with acetic acid to neutrality at 0-5xc2x0 C. and dilution with water to precipitate dihydroartemisinin), A. Brossi, et al., Journal of Medicinal Chemistry, 31:645-650 (1988). Using dihydroartemisinin as a starting compound a large number of other derivatives, such as, 
1 R=H
2 R=CH3 
3 R=CH2CH3 
4 R=COCH2CH2COONa
5 R=CH2C6H4COOH
6 R=CH2C6H4COONa
7 
artemether (compound II-2), arteether (II-3), sodium artesunate (II-4), artelinic acid (II-5), soduim artelinate (II-6), dihydroartemisinin condensation by-product (II-7) and the olefinic compound structure III, 
have been produced.
Artemether (II-2) is produced by reacting E-dihydroartemisinin with boron trifluoride (BF3) etherate or HCl in methanol:benzene (1:2) at room temperature. A mixture of E-and xcex94-artemether (70:30) is obtained, from which the former is isolated by colunm chromatography and recrystallized from hexane or methanol, R. Haynes, Transactions of the Royal Society of Tropical Medicine and Hygiene, 88(1): S1/23-S1/26 (1994). For arteether (II-3), (Brossi, et al., 1988), the xcex94-isomer is equilibrated (epimerized) to the E-isomer in ethanol:benzene mixture containing BF3 etherate. Treatment of dihydroartemisinin with an unspecified dehydrating agent yields both the olefinic compound, (III), and the dihydroartemisinin condensation by-product (II-7), formed on addition of dihydroartemisinin to (III), M. Cao, et al., Chem. Abstr., 100:34720k (1984). Until recently, the secondary hydroxy group in dihydroartemisinin (II-1) provided the only site in an active artemisinin-related compound that had been used for derivatization. See B. Venugopalan, xe2x80x9cSynthesis of a Novel Ring Contracted Artemisinin Derivative,xe2x80x9d Bioorganic and Medicinal Chemistry Letters, 4(5):751-752 (1994).
The potency of various artemisinin-derivatives in comparison to artemisinin as a function of the concentration at which the parasitemia is 90 percent suppressed (SD90) was reported by D. L. Klayman, xe2x80x9cQinghaosu (Artemisinin): An Antimalarial Drug from China,xe2x80x9d Science 228:1049-1055 (1985). Dr. Klayman reported that the olefinic compound III is inactive against P. berghei-infected mice, whereas the dihydroartemisinin condensation by-product (II-7) has an SD90 of 10 mg/Kg in P. berghei-infected mice. Thus, the dihydroartemisinin ether dimer proved to be less potent than artemisinin, which has an SD90 of 6.20 mg/Kg. Following, in order of their overall antimalarial efficacy, are the three types of derivatives of dihydroartemisinin (II-1) that have been produced: (artemisinin) less than ethers (II, R=alkyl) less than esters [II,=C(xe2x95x90O)-alkyl or -aryl] less than carbonates [II, R=C (xe2x95x90O)O-alkyl or -aryl].
Other rational design of structurally simpler analogs of artemisinin has led to the synthesis of various trioxanes, some of which possess excellent antimalarial activity. Posner, G. H., et al., reported the chemistry and biology of a series of new structurally simple, easily prepared, racemic 1,2,4-trioxanes (disclosed in U.S. Pat. No. 5,225,437 and incorporated herein by reference) that are tricyclic (lacking the lactone ring present in tetracyclic artemisinin I) and that are derivatives of trioxane alcohol IV. 
having the relative stereochemistry shown above. Especially attractive features of trioxane alcohol IV are the following: (1) its straightforward and easy preparation from cheap and readily available starting materials, (2) its availability on gram scale, and (3) its easy one-step conversion, using standard chemical transformations, into alcohol derivatives such as esters and ethers, without destruction of the crucial trioxane framework. See, Posner, G. H., et al., J Med Chem., 35:2459-2467 (1992), incorporated herein by reference. The complete chemical synthesis of artemisinin and a variety of other derivatives is reviewed by Sharma, R. P., et al., Heterocycles, 32(8):1593-1638 (1991), and is incorporated herein by reference.
Metabolic studies by Baker, et al., demonstrated that B-arteether (II-3), one of the antimalarial derivatives discussed previously, was rapidly converted by rat liver microsomes into dihydroartemisinin (II-1). See Baker, J. K., et al., Biol. Mass Spect., 20:609-628 (1991). This finding and the fact that the most effective artemisinin derivatives against malaria have been ethers or esters of (II-1) suggest that they were prodrugs for (II-1). The controlled slow formation of (II-1), however, is not desirable in view of its short half-life in plasma (less than two hours) and relatively high toxicity.
The successful synthesis of anticancer and antiviral drugs by replacing a carbon-nitrogen bond in nucleosides by a carbon-carbon bond (C-nucleosides) prompted the preparation of several 10-alkyldeoxoartemisinins, V, 
wherein R is 1-allyl, propyl, methyl, or ethyl. Typically, these syntheses involved five or six steps and the reported yields were only about 12 percent. See, Jung, M., et al., Synlett., 743-744 (1990); and Haynes, R. K., et al, SynIett., 481-484 (1992).
Heterolytic cleavage of the peroxide Oxe2x80x94O bond via SN2 attack of nucleophiles is well documented., see Adam, W. et al, J Am. Chem. Soc., 114:5591 (1992) and Razuvaev, G. A., et al, T. G. In Organic Peroxides; D. Swem, Ed., John Wiley and Sons, New York, 3:141-270, (1972). For example, tert-butyl ethers are conveniently prepared by Grignard nucleophilic attack on the Oxe2x80x94O bond in tert-butyl peresters, see Lawesson, S.-O., et al., J. Am. Chem. Soc., 81:4230, (1959). Also, 3,3-disubstituted-1,2-dioxetanes react with organolithium reagents primarily via SN2 Oxe2x80x94O bond cleavage (with regioselective attack at the sterically less encumbered O atom) to form E-hydroxy ethers (Adam, W., et al., Chem. Ber, 125:235, (1992)) and bicyclic endoperoxides likewise react with lithium and magnesium organometallics to produce Oxe2x80x94O bond-cleaved hydroxy ethers. See, Schwaebe, M. K., et al, Tetrahedron Lett., 37:6635 (1996). When a dialkyl peroxide Oxe2x80x94O bond is sterically hindered, then nucleophilic attack by a reactive organometallic reagent is made more difficult; an excellent example of this phenomenon leading to chemoselective nucleophilic addition of an organolithium reagent to the aldehyde carbonyl group in a peroxy aldehyde. See, Dussault, P., et al., T. J. Org. Chem., 58:5469 (1993) and Dussault, P., Synlett, 997 (1995). 1,2,4-Trioxanes in the artemisinin family undergo peroxide Oxe2x80x94O bond cleavage when exposed to dimethylcopperlithium and to trityllithium; in these two cases, however, single-electron-reductive cleavage of the peroxide bond is likely occurring. See, Posner, G. H. et al., J Am. Chem. Soc., 114:8328 (1992). Sodium borohydride chemoselectively reduces artemisinin (I) into its lactol (II-1), but more potent lithium aluminum hydride reduces both the lactone carbonyl group and the trioxane Oxe2x80x94O bond. See, Wu, Y, et al, Youji Huaxue, 153, (1986); Chem. Abstr. 1986, 105, 191426n.
Based on these published precedents, it seemed that it would be very difficult to find any reactive organometallic reagents that would add chemoselectively to the lactone carbonyl group (less electrophilic than an aldehyde) of trioxane lactone artemisinin (I) without also cleaving the trioxane Oxe2x80x94O bond. In fact, exposing artemisinin to 1.2 equivalent of phenyllithium in THF at xe2x88x9278xc2x0 C. produced at least three major products (not characterized).
Over the past thirty years only a few drugs isolated from higher plants have yielded clinical agents, the outstanding examples being vinblastine and vincristine from the Madagascan periwinkle, Catharanthus roseus, etoposide, the semi-synthetic lignan, from Mayapple Podophyllum peltatum and the diterpenoid taxol, commonly referred to as paclitaxel, from the Pacific yew, Taxus brevifolia. Of these agents, paclitaxel is the most exciting, recently receiving approval by the Food and Drug Administration for the treatment of refractory ovarian cancer. Since the isolation of artemisinin, there has been a concerted effort by investigators to study other therapeutic applications of artemisinin and its derivatives.
National Institutes of Health reported that artemisinin is inactive against P388 leukemia. See NCI Report on NSC 369397 (tested on Oct. 25, 1983). Later anticancer studies that have been conducted on cell line panels consisting of 60 lines organized into nine, disease-related subpanels including leukemia, non-small-cell lung cancer, colon, CNS, melanoma, ovarian, renal, prostate and breast cancers, further confirm that artemisinin displays very little anticancer activity. A series of artemisinin-related endoperoxides were tested for cytotoxicity to Ehrlich ascites tumor (EAT) cells using the microculture tetrazolum (MTT) assay, H. J. Woerdenbag, et al., xe2x80x9cCytotoxicity of Artemisinin-Related Endoperoxides to Ehrlich Ascites Tumor Cells,xe2x80x9d Journal of Natural Products, 56(6):849-856 (1993). The MTT assay, used to test the artemisinin-related endoperoxides for cytotoxicity, is based on the metabolic reduction of soluble tetrazolium salts into insoluble colored formazan products by mitochondrial dehydrogenase activity of the tumor cells. As parameters for cytotoxicity, the IC50 and IC80 values, the drug concentrations causing respectively 50% and 80% growth inhibition of the tumor cells, were used. Artemisinin (I) had an IC50 value of 29.8 Π M. Derivatives of dihydroartemisinin (II-1) being developed as antimalarial drugs (artemether (II-2), arteether (III-3), sodium artesunate (II-4), artelinic acid (II-5) and sodium artelinate (II-6)), exhibited a somewhat more potent cytotoxicity. Their IC50 values ranged from 12.2 Π M to 19.9 Π M. The dihydroartemisinin condensation by-product dimer (II-7), disclosed previously by M. Cao, et al., 1984, was the most potent cytotoxic agent, its IC50 being 1.4 Π M. At this drug concentration the condensation by-product (II-7) is approximately twenty-two times more cytotoxic than artemisinin and sixty times more cytotoxic than dihydroartemisinin (II-1), the parent compound.
While artemisinin and its related derivatives (II1-6) discussed above demonstrated zero to slight antiproliferative and antitumor activity, it has been discovered that a class of artemisinin dimer compounds exhibits antiproliferative and antitumor activities that are, in vitro, equivalent to or greater than known antiproliferative and antitumor agents. See, U.S. Pat. No. 5,677,468 incorporated herein by reference. Unfortunately, while the in vitro results of these artemisinin compounds are encouraging these compounds do not appear to have significant antitumor activity on the treatment of tumor cells in mice.
There is still a need, therefore, to develop methods for the formation of hydrolytically stable C-10 carbon-substituted artemisinin compounds and structural analogs thereof having antimalarial, and antiproliferative and antitumor activities that are equivalent to or greater than those of known antimalarial, and antiproliferative and antitumor agents, respectively, wherein the method does not result in cleavage of the trioxane Oxe2x80x94O bond.
Accordingly, this invention provides a class of artemisinin related dimers which demonstrate antiproliferative and antitumor activities.
More specifically, this invention provides a class of trioxane dimers which demonstrate antiproliferative and antitumor activities and that are considerably more stable than artemether and related C-10 ethers and esters toward hydrolysis.
This invention further provides artemisinin dimers to be used clinically as chemotherapeutic anticancer drugs.
This invention further provides a class of trioxane monomers to be used clinically as chemotherapeutic antimalarial drugs and methods of producing the same.
Additional objects, advantages and novel features of this invention shall be set forth in part in the description and examples that follow, and in part will become apparent to those skilled in the art upon examination of the following specification or may be learned by the practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities, combinations, compositions, and methods particularly pointed out in the appended claims.
To achieve the foregoing and other objects and in accordance with the purposes of the present invention, as embodied and broadly described therein the compositions of this invention comprise C-10 carbon-substituted derivatives of the trioxane exocyclic alkene (VII) of the following structure 
or diastereomers thereof, having antimalarial, and antiproliferative and antitumor activities wherein, the monomers of the present invention are formed when n is 1 and R is alkyl, cycloalkyl, heteroalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl or heteroaryl. The term xe2x80x9calkylxe2x80x9d includes straight chain or branched alkyl compounds comprising 1-20 carbon atoms and cyclic alkyl compounds comprising 5-10 carbon atoms. The term xe2x80x9cheteroalkylxe2x80x9d includes polyalkylene glycols such as polyethylene glycol (PEG). The term xe2x80x9carylxe2x80x9d means a phenyl or phenyl group substituted by 1 or more substituents selected from the group comprising halogen, nitro, amino, hydroxy, thiohydroxy, lower alkoxy, lower thioalkyl, lower alkyl, NHC(xe2x95x90O)R wherein R is aryl or lower alkyl, COOH, or COOR2 wherein R2 is aryl or lower alkyl. The term xe2x80x9cheteroarylxe2x80x9d includes 5 or 6 membered heteroaromatic rings comprising one or more heteroatoms selected from N, O or S, unsubstituted or substituted by halogen, nitro, amino, hydroxy, thiohydroxy, lower alkoxy, lower alkyl, NHC(xe2x95x90O)R1 wherein R is aryl or lower alkyl, COOH, or COOR2 wherein R2 is aryl or lower alkyl. The term xe2x80x9clower alkylxe2x80x9d means straight or branched hydrocarbon radicals comprising 1-20 carbon atoms and include, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, n-pentyl and the like. xe2x80x9cHalogenxe2x80x9d is fluorine, chlorine, bromine or iodine.
The dimers of the present invention are formed when n is 2 and R is a linker including, but not limited to, alkyl, cycloalkyl, heteroalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl or heteroaryl. The term xe2x80x9calkylxe2x80x9d includes straight chain or branched bivalent alkyl compounds comprising 1-20 carbon atoms and cyclic alkyl compounds comprising 5-10 carbon atoms. The term xe2x80x9cheteroalkylxe2x80x9d includes bivalent polyalkylene glycols such as polyethylene glycol (PEG). The term xe2x80x9carylxe2x80x9d means a bivalent phenyl or phenyl group substituted by 1 or more substituents selected from the group comprising halogen, nitro, amino, hydroxy, thiohydroxy, lower alkoxy, lower thioalkyl, lower alkyl, NHC(xe2x95x90O)R1 wherein R1 is aryl or lower alkyl, COOH, or COOR2 wherein R2 is aryl or lower alkyl. The term xe2x80x9cheteroarylxe2x80x9d includes bivalent 5 or 6 membered heteroaromatic rings comprising one or more heteroatoms selected from N, O or S, unsubstituted or substituted by halogen, nitro, amino, hydroxy, thiohydroxy, lower alkoxy, lower alkyl, NHC(xe2x95x90O)R1 wherein R1 is aryl or lower alkyl, COOH, or COOR2 wherein R2 is aryl or lower alkyl. The term xe2x80x9clower alkylxe2x80x9d means straight or branched hydrocarbon radicals comprising 1-20 carbon atoms and include, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, n-pentyl and the like. xe2x80x9cHalogenxe2x80x9d is fluorine, chlorine, bromine or iodine.